Intravital imaging of cardiac function at the single-cell level.

Knowledge of cardiomyocyte biology is limited by the lack of methods to interrogate single-cell physiology in vivo. Here we show that contracting myocytes can indeed be imaged with optical microscopy at high temporal and spatial resolution in the beating murine heart, allowing visualization of individual sarcomeres and measurement of the single cardiomyocyte contractile cycle. Collectively, this has been enabled by efficient tissue stabilization, a prospective real-time cardiac gating approach, an image processing algorithm for motion-artifact-free imaging throughout the cardiac cycle, and a fluorescent membrane staining protocol. Quantification of cardiomyocyte contractile function in vivo opens many possibilities for investigating myocardial disease and therapeutic intervention at the cellular level.

Differential contribution of monocytes to heart macrophages in steady-state and after myocardial infarction.

RATIONALE: Macrophages populate the steady-state myocardium. Previously, all macrophages were thought to arise from monocytes; however, it emerged that, in several organs, tissue-resident macrophages may self-maintain through local proliferation. OBJECTIVE: Our aim was to study the contribution of monocytes to cardiac-resident macrophages in steady state, after macrophage depletion in CD11b(DTR/+) mice and in myocardial infarction. METHODS AND RESULTS: Using in vivo fate mapping and flow cytometry, we estimated that during steady state the heart macrophage population turns over in ≈1 month. To explore the source of cardiac-resident macrophages, we joined the circulation of mice using parabiosis. After 6 weeks, we observed blood monocyte chimerism of 35.3±3.4%, whereas heart macrophages showed a much lower chimerism of 2.7±0.5% (P<0.01). Macrophages self-renewed locally through proliferation: 2.1±0.3% incorporated bromodeoxyuridine 2 hours after a single injection, and 13.7±1.4% heart macrophages stained positive for the cell cycle marker Ki-67. The cells likely participate in defense against infection, because we found them to ingest fluorescently labeled bacteria. In ischemic myocardium, we observed that tissue-resident macrophages died locally, whereas some also migrated to hematopoietic organs. If the steady state was perturbed by coronary ligation or diphtheria toxin-induced macrophage depletion in CD11b(DTR/+) mice, blood monocytes replenished heart macrophages. However, in the chronic phase after myocardial infarction, macrophages residing in the infarct were again independent from the blood monocyte pool, returning to the steady-state situation. CONCLUSIONS: In this study, we show differential contribution of monocytes to heart macrophages during steady state, after macrophage depletion or in the acute and chronic phase after myocardial infarction. We found that macrophages participate in the immunosurveillance of myocardial tissue. These data correspond with previous studies on tissue-resident macrophages and raise important questions on the fate and function of macrophages during the development of heart failure.

Monocyte-directed RNAi targeting CCR2 improves infarct healing in atherosclerosis-prone mice.

BACKGROUND: Exaggerated and prolonged inflammation after myocardial infarction (MI) accelerates left ventricular remodeling. Inflammatory pathways may present a therapeutic target to prevent post-MI heart failure. However, the appropriate magnitude and timing of interventions are largely unknown, in part because noninvasive monitoring tools are lacking. Here, we used nanoparticle-facilitated silencing of CCR2, the chemokine receptor that governs inflammatory Ly-6C(high) monocyte subset traffic, to reduce infarct inflammation in apolipoprotein E-deficient (apoE(-/-)) mice after MI. We used dual-target positron emission tomography/magnetic resonance imaging of transglutaminase factor XIII (FXIII) and myeloperoxidase (MPO) activity to monitor how monocyte subset-targeted RNAi altered infarct inflammation and healing. METHODS AND RESULTS: Flow cytometry, gene expression analysis, and histology revealed reduced monocyte numbers and enhanced resolution of inflammation in infarcted hearts of apoE(-/-) mice that were treated with nanoparticle-encapsulated siRNA. To follow extracellular matrix cross-linking noninvasively, we developed a fluorine-18-labeled positron emission tomography agent ((18)F-FXIII). Recruitment of MPO-rich inflammatory leukocytes was imaged with a molecular magnetic resonance imaging sensor of MPO activity (MPO-Gd). Positron emission tomography/magnetic resonance imaging detected anti-inflammatory effects of intravenous nanoparticle-facilitated siRNA therapy (75% decrease of MPO-Gd signal; P<0.05), whereas (18)F-FXIII positron emission tomography reflected unimpeded matrix cross-linking in the infarct. Silencing of CCR2 during the first week after MI improved ejection fraction on day 21 after MI from 29% to 35% (P<0.05). CONCLUSION: CCR2-targeted RNAi reduced recruitment of Ly-6C(high) monocytes, attenuated infarct inflammation, and curbed post-MI left ventricular remodeling.

Fluorescent exendin-4 derivatives for pancreatic ß-cell analysis.

The ability to reliably identify pancreatic ß-cells would have far reaching implications for a greater understanding of ß-cell biology, measurement of ß-cell mass in diabetes, islet transplantation, and drug development. The glucagon-like peptide-1 receptor (GLP1R) is highly expressed on the surface of insulin producing pancreatic ß-cells. Using systematic modifications of the GLP1R ligand, exendin-4, we screened over 25 compounds and identified a palette of fluorescent exendin-4 with high GLP1R binding affinity. We show considerable differences in affinity, as well as utility of the top candidates for flow cytometry and microscopy of ß-cells. Some of the developed compounds should be particularly useful for basic and translational ß-cell research.

Comparison of select cancer biomarkers in human circulating and bulk tumor cells using magnetic nanoparticles and a miniaturized micro-NMR system.

Circulating tumor cells (CTC) harvested from peripheral blood have received significant interest as sources for serial sampling to gauge treatment efficacy. Nanotechnology and microfluidic based approaches are emerging to facilitate such analyses. While of considerable clinical importance, there is little information on how similar or different CTCs are from their shedding bulk tumors. In this clinical study, paired tumor fine needle aspirate and peripheral blood samples were obtained from cancer patients during image-guided biopsy. Using targeted magnetic nanoparticles and a point-of-care micro-NMR system, we compared selected biomarkers (EpCAM, EGFR, HER-2 and vimentin) in both CTC and fine needle biopsies of solid epithelial cancers. We show a weak correlation between each paired sample, suggesting that use of CTC as "liquid biopsies" and proxies to metastatic solid lesions could be misleading. FROM THE CLINICAL EDITOR: In this clinical study, paired tumor fine needle aspirate and peripheral blood samples were obtained from patients with solid epithelial cancers during image-guided biopsy. Using targeted magnetic nanoparticles and a point-of-care micro-NMR system, the authors compared selected biomarkers in both circulating tumor cells (CTC) and fine needle biopsies, demonstrating a weak correlation between each paired sample, suggesting that use of CTC could be misleading in this context.